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Expression, purification and characterization of the HIV-1 coreceptor CCR5 and its ligand RANTES and high-pressure NMR investigation of hydrogen bonds in biomolecules

Nisius, Lydia. Expression, purification and characterization of the HIV-1 coreceptor CCR5 and its ligand RANTES and high-pressure NMR investigation of hydrogen bonds in biomolecules. 2010, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9251

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Abstract

Nuclear magnetic resonance spectroscopy is a very versatile technique for the investigation of biomolecular structures and dynamics. In this thesis, solution and solid state NMR methods have been applied to study a membrane protein and its interactions with a ligand and an antagonist, to characterize the thermodynamics of protein dimerization, and to observe changes in the hydrogen bond network of proteins and nucleic acids under the influence of high pressure.
The first part describes the expression, purification, and characterization of the membrane protein CCR5 and its ligand RANTES. The second part is concerned with the effect of pressure on the hydrogen bond network in proteins and nucleic acids.
The G protein-coupled receptor CCR5 is the major HIV-1 coreceptor and thus a very important target for the development of HIV-1 entry inhibitors. The large-scale expression and purification of CCR5, leading to 1 mg of pure protein per liter cell culture after purification, is described. The receptor's functionality is shown via recognition by a conformation-dependent antibody and by binding of the endogenous ligand RANTES.
The possibility to obtain large amounts of functional CCR5 overcomes a major bottleneck for the structural characterization of this membrane protein.
The receptor and its ligand were further characterized by NMR spectroscopy.
First heteronuclear solution state NMR spectra of selectively labeled CCR5 were obtained.
The interaction of the receptor with the chemokine RANTES was studied by solution NMR as well as in a lipid environment using solid state NMR experiments. In these experiments, binding of RANTES to CCR5 is observed.
Many chemokines undergo dimerization in the low micro- to millimolar range, but the biological relevance of this phenomenon is under debate. The temperature and pressure dependence of the RANTES monomer/dimer equilibrium was characterized by NMR spectroscopy. This study reveals the enthalpy and entropy contributions to RANTES dimerization. In addition, a pressure-induced unfolded state was observed for the first time.
A solely monomeric mutant of RANTES was designed and shown to inhibit HIV-1 infection in vitro. This is the basis for further studies of the complex between RANTES and CCR5 as well as CCR5-derived peptides.
Hydrogen bonds are extremely important to stabilize the structures of proteins and nucleic acids. In this thesis, methods to measure the scalar couplings across hydrogen bonds in these biomolecules are described. They were applied to study pressure-dependent changes in the hydrogen bond scalar couplings in ubiquitin. The most pressure-labile regions of the protein were identified.
These largely correspond to regions which are destabilized at elevated temperature.
Pressure- and temperature-dependent hydrogen bond scalar couplings have also been measured in a small RNA hairpin. The data indicate that the average pressure-induced changes of hydrogen bond length are similar in nucleic acids and proteins.
Advisors:Grzesiek, Stephan
Committee Members:Engel, Andreas
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
Item Type:Thesis
Thesis no:9251
Bibsysno:Link to catalogue
Number of Pages:118 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:29 Dec 2010 09:57

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